In this application, we seek (l) to extend our use of forward genetics in mammalian cells to new investigations of signaling pathways and (2) to exploit findings made with STAT I-null cells that have defined new aspects of gene regulation by this ubiquitous transcription factor. Specific aim l. Mutant cell lines unresponsive to TNFalpha. We have developed a new selection scheme in which the survival of mutant cells that fail to respond to a signal depends on their resistance to gancyclovir, due to their failure to induce Herpes thyrnidine kinase. This scheme represents a marked improvement over our previous method, which employed guanine phosphoribosyl transferase and 6-thioguanine, because low basal expression of the selectable marker can now be tolerated, greatly extending the range of experimental systems that can be investigated. Using gancyclovir to select cells that fail to respond to IL-1 or TNF-alpha, we have already obtained many independent mutant cell lines, and we propose here to continue the isolation, characterization and complementation of mutants that fail to respond to TNF-alpha. This study will add genetic analysis to the growing biochemical definition of this important pathway. Specific aim 2. Novel aspects of STAT1. A. STAT1 and constitutive gene expression. STAT1 is essential for efficient constitutive expression of caspases 1-3 and other genes through mechanisms that do not involve the activation of STAT1 through tyrosine phosphorylation or dimer formation. We will define the DNA elements that respond to this novel functional form of STAT l and search for putative transcription factor partners. We will also use differential display technology to identify additional genes that require STAT1, or STAT2, for constitutive expression. B. STAT1 dimers as negative regulators. We have found that expression of the c-myc gene is negatively regulated by IFN-gamma through the formation of STAT1 dimers. In normal cells, cotreatment with IFN-gamma antagonizes the induction of c-myc by PDGF. We will determine whether STAT1 dimers effect negative regulation of expression through a consensus GAS element in the c-myc promoter or through other elements and investigate the domains of STAT1 required for negative regulation. C. STAT1- independent responses to IFN-gamma. Remarkably, in STATl -null cells, treatment with IFN-gamma alone stimulates c-myc expression, revealing the existence of an unknown pathway for IFN-gamma-mediated signaling. This novel activity will be defined and the mechanism will be explored by using forward genetics. Through the work proposed in this application we will develop better ways to obtain and complement mutant cell lines and will contribute to the analysis of several important signaling pathways.